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• W2093034951 abstract "Spider venom is a complex mixture of bioactive peptides to subdue their prey. Early estimates suggested that over 400 venom peptides are produced per species. In order to investigate the mechanisms responsible for this impressive diversity, transcriptomics based on second generation high throughput sequencing was combined with peptidomic assays to characterize the venom of the tarantula Haplopelma hainanum. The genes expressed in the venom glands were identified, and the bioactivity of their protein products was analyzed using the patch clamp technique. A total of 1,136 potential toxin precursors were identified that clustered into 90 toxin groups, of which 72 were novel. The toxin peptides clustered into 20 cysteine scaffolds that included between 4 and 12 cysteines, and 14 of these groups were newly identified in this spider. Highly abundant toxin peptide transcripts were present and resulted from hypermutation and/or fragment insertion/deletion. In combination with variable post-translational modifications, this genetic variability explained how a limited set of genes can generate hundreds of toxin peptides in venom glands. Furthermore, the intraspecies venom variability illustrated the dynamic nature of spider venom and revealed how complex components work together to generate diverse bioactivities that facilitate adaptation to changing environments, types of prey, and milking regimes in captivity.Background: Spider venom is a complex mixture and contains over 400 venom peptides/species.Results: Highly abundant hypermutation, fragment insertion/deletion, and variable post-translational modifications were observed in venom gland, and the highly diverse toxins exhibit diverse functions.Conclusion: Spider toxins are abundant and variable, factors that work together to generate diverse bioactivities.Significance: Our study reveals the mechanism of structural and functional diversity of peptide toxins from Ornithoctonus hainana. Spider venom is a complex mixture of bioactive peptides to subdue their prey. Early estimates suggested that over 400 venom peptides are produced per species. In order to investigate the mechanisms responsible for this impressive diversity, transcriptomics based on second generation high throughput sequencing was combined with peptidomic assays to characterize the venom of the tarantula Haplopelma hainanum. The genes expressed in the venom glands were identified, and the bioactivity of their protein products was analyzed using the patch clamp technique. A total of 1,136 potential toxin precursors were identified that clustered into 90 toxin groups, of which 72 were novel. The toxin peptides clustered into 20 cysteine scaffolds that included between 4 and 12 cysteines, and 14 of these groups were newly identified in this spider. Highly abundant toxin peptide transcripts were present and resulted from hypermutation and/or fragment insertion/deletion. In combination with variable post-translational modifications, this genetic variability explained how a limited set of genes can generate hundreds of toxin peptides in venom glands. Furthermore, the intraspecies venom variability illustrated the dynamic nature of spider venom and revealed how complex components work together to generate diverse bioactivities that facilitate adaptation to changing environments, types of prey, and milking regimes in captivity. Background: Spider venom is a complex mixture and contains over 400 venom peptides/species. Results: Highly abundant hypermutation, fragment insertion/deletion, and variable post-translational modifications were observed in venom gland, and the highly diverse toxins exhibit diverse functions. Conclusion: Spider toxins are abundant and variable, factors that work together to generate diverse bioactivities. Significance: Our study reveals the mechanism of structural and functional diversity of peptide toxins from Ornithoctonus hainana. Structural and functional diversity of peptide toxins from tarantula Haplopelma hainanum (Ornithoctonus hainana) venom revealed by transcriptomic, peptidomic, and patch clamp approaches.Journal of Biological ChemistryVol. 290Issue 44PreviewVOLUME 290 (2015) PAGES 14192–14207 Full-Text PDF Open Access Spiders are some of the most ancient venomous animals and have been roaming the earth for 300 million years, numbering nearly 40,000 species. Tarantulas are a group of often hairy and large arachnids that comprise more than 860 species. Like other venomous animals, these predators use a complex arsenal of venom to paralyze and kill their prey, and many of these toxins have proved to be invaluable tools for pharmacological studies of voltage-sensitive and ligand-gated ion channels (1.Binford G.J. Bodner M.R. Cordes M.H. Baldwin K.L. Rynerson M.R. Burns S.N. Zobel-Thropp P.A. Molecular evolution, functional variation, and proposed nomenclature of the gene family that includes sphingomyelinase D in sicariid spider venoms.Mol. Biol. Evol. 2009; 26: 547-566Crossref PubMed Scopus (87) Google Scholar2.Liang S. Proteome and peptidome profiling of spider venoms.Expert Rev. Proteomics. 2008; 5: 731-746Crossref PubMed Scopus (49) Google Scholar, 3.Grishin E. Polypeptide neurotoxins from spider venoms.Eur. J. Biochem. 1999; 264: 276-280Crossref PubMed Scopus (83) Google Scholar4.Liang S. An overview of peptide toxins from the venom of the Chinese bird spider Selenocosmia huwena Wang [=Ornithoctonus huwena (Wang)].Toxicon. 2004; 43: 575-585Crossref PubMed Scopus (107) Google Scholar). The vast majority of spider toxins are cysteine-rich polypeptides, and their properties and structures have been reviewed in detail (4.Liang S. An overview of peptide toxins from the venom of the Chinese bird spider Selenocosmia huwena Wang [=Ornithoctonus huwena (Wang)].Toxicon. 2004; 43: 575-585Crossref PubMed Scopus (107) Google Scholar5.Escoubas P. Rash L. Tarantulas: eight-legged pharmacists and combinatorial chemists.Toxicon. 2004; 43: 555-574Crossref PubMed Scopus (174) Google Scholar, 6.Corzo G. Escoubas P. Pharmacologically active spider peptide toxins.Cell Mol. Life Sci. 2003; 60: 2409-2426Crossref PubMed Scopus (72) Google Scholar7.Rash L.D. Hodgson W.C. Pharmacology and biochemistry of spider venoms.Toxicon. 2002; 40: 225-254Crossref PubMed Scopus (284) Google Scholar). The molecular diversity of spider toxins has also been investigated, and they appear to be based on a limited set of structural scaffolds, such as the inhibitor cysteine knot (ICK) 4The abbreviations used are: ICKinhibitor cysteine knotTTXtetrodotoxinTTX-Stetrodotoxin-sensitiveDRGdorsal root ganglionESTexpressed sequence tagcontiggroup of overlapping clonesGOgene ontologyVGSCvoltage-gated sodium channel. and disulfide-directed β-hairpin, whose cysteines form 3–5 characteristic disulfide bonds, which is a much smaller number than toxins from other species, such as marine cone snails. The lack of a complete genome sequence may be responsible for our currently limited knowledge of the cysteine pattern diversity present in tarantula toxins, and improving this knowledge will be challenging. inhibitor cysteine knot tetrodotoxin tetrodotoxin-sensitive dorsal root ganglion expressed sequence tag group of overlapping clones gene ontology voltage-gated sodium channel. Expressed sequence tags (ESTs) are short sequence reads derived from cDNA libraries that are useful tools for the identification of transcripts in species without a fully sequenced genome (8.Fernandes-Pedrosa Mde F. Junqueira-de-Azevedo Ide L. Gonc̃alves-de-Andrade R.M. Kobashi L.S. Almeida D.D. Ho P.L. Tambourgi D.V. Transcriptome analysis of Loxosceles laeta (Araneae, Sicariidae) spider venomous gland using expressed sequence tags.BMC Genomics. 2008; 9: 279Crossref PubMed Scopus (96) Google Scholar, 9.Chen J. Zhao L. Jiang L. Meng E. Zhang Y. Xiong X. Liang S. Transcriptome analysis revealed novel possible venom components and cellular processes of the tarantula Chilobrachys jingzhao venom gland.Toxicon. 2008; 52: 794-806Crossref PubMed Scopus (36) Google Scholar). In the past few years, all reports on tarantula toxin transcriptomics have utilized classical cloning and Sanger sequencing strategies (9.Chen J. Zhao L. Jiang L. Meng E. Zhang Y. Xiong X. Liang S. Transcriptome analysis revealed novel possible venom components and cellular processes of the tarantula Chilobrachys jingzhao venom gland.Toxicon. 2008; 52: 794-806Crossref PubMed Scopus (36) Google Scholar). In previous work, 420 peptides were detected by mass spectrometry, but few could be paired with peptide precursors identified from cDNA and genomic DNA sequencing (10.Tang X. Zhang Y. Hu W. Xu D. Tao H. Yang X. Li Y. Jiang L. Liang S. Molecular diversification of peptide toxins from the tarantula Haplopelma hainanum (Ornithoctonus hainana) venom based on transcriptomic, peptidomic, and genomic analyses.J. Proteome Res. 2010; 9: 2550-2564Crossref PubMed Scopus (83) Google Scholar). Furthermore, this limited data focused mainly on highly abundant and smaller toxin precursors, whereas less prevalent and longer gene sequences were largely ignored, which has proved a barrier to research on the molecular diversity and genetic mechanisms of toxin evolution (11.Escoubas P. Molecular diversification in spider venoms: a web of combinatorial peptide libraries.Mol. Divers. 2006; 10: 545-554Crossref PubMed Scopus (86) Google Scholar). The relatively new 454 Life Sciences pyrosequencing technology has been successfully implemented in a number of species, including spiders (12.Durban J. Juárez P. Angulo Y. Lomonte B. Flores-Diaz M. Alape-Girón A. Sasa M. Sanz L. Gutiérrez J.M. Dopazo J. Conesa A. Calvete J.J. Profiling the venom gland transcriptomes of Costa Rican snakes by 454 pyrosequencing.BMC Genomics. 2011; 12: 259Crossref PubMed Scopus (86) Google Scholar13.Terrat Y. Biass D. Dutertre S. Favreau P. Remm M. Stöcklin R. Piquemal D. Ducancel F. High-resolution picture of a venom gland transcriptome: case study with the marine snail Conus consors.Toxicon. 2012; 59: 34-46Crossref PubMed Scopus (73) Google Scholar, 14.Rokyta D.R. Lemmon A.R. Margres M.J. Aronow K. The venom-gland transcriptome of the eastern diamondback rattlesnake (Crotalus adamanteus).BMC Genomics. 2012; 13: 312Crossref PubMed Scopus (211) Google Scholar, 15.Lluisma A.O. Milash B.A. Moore B. Olivera B.M. Bandyopadhyay P.K. Novel venom peptides from the cone snail Conus pulicarius discovered through next-generation sequencing of its venom duct transcriptome.Mar. Genomics. 2012; 5: 43-51Crossref PubMed Scopus (58) Google Scholar16.Prosdocimi F. Bittencourt D. da Silva F.R. Kirst M. Motta P.C. Rech E.L. Spinning gland transcriptomics from two main clades of spiders (order: Araneae): insights on their molecular, anatomical and behavioral evolution.PLoS One. 2011; 6: e21634Crossref PubMed Scopus (27) Google Scholar). This approach provides a more comprehensive landscape of the transcriptomic content of venom glands and has improved technical capabilities that identify longer sequences, a wider range of sensitivity, and greater accuracy than traditional Sanger sequencing (17.Zhang J. Chiodini R. Badr A. Zhang G. The impact of next-generation sequencing on genomics.J. Genet. Genomics. 2011; 38: 95-109Crossref PubMed Scopus (352) Google Scholar, 18.Liu L. Li Y. Li S. Hu N. He Y. Pong R. Lin D. Lu L. Law M. Comparison of next-generation sequencing systems.J. Biomed. Biotechnol. 2012; 2012: 251364Crossref PubMed Scopus (966) Google Scholar). The longer reads (>300 bp on average) generated with 454 pyrosequencing allow coverage of full-length (60–120 amino acids) precursors, which affords the direct identification of spider toxin precursors and avoids the errors inherent in assembling reads into contigs as typically required for other second generation technologies that generate shorter read lengths (19.Zheng Y. Zhao L. Gao J. Fei Z. iAssembler: a package for de novo assembly of Roche-454/Sanger transcriptome sequences.BMC Bioinformatics. 2011; 12: 453Crossref PubMed Scopus (112) Google Scholar). In this study, 1,136 toxin precursors were identified using 454 Life Sciences pyrosequencing and classified into 15 classes (class A–O). Sequence analysis revealed that extensive hypermutation and fragment insertion/deletion dramatically increased the molecular diversity of toxin transcripts, and this diversity was further enhanced by highly variable post-translational modifications. The high intraspecies variability may explain the transcriptome differences between this work and previous work. Analysis of the bioactivity of 14 of the identified toxins against DRG ion channels using patch clamping revealed functional diversity. In conclusion, variable peptide processing and selective expression explain how a limited set of gene transcripts can generate hundreds of toxin peptides in spider venom that have diverse activities and cooperate to subdue potential prey species. Dithiothreitol (DTT), iodoacetamide, and trifluoroacetic acid (TFA) were obtained from Sigma. Acetonitrile was a domestic product (chromatogram grade). Milli-Q H2O was used for the preparation of all buffers. Other chemicals were of analytical grade. The tarantula spiders were collected from Hainan, China. Venom glands of Haplopelma hainanum were obtained 2 days after being milked by electrical stimulation and were ground to fine powder in liquid nitrogen. Total RNA was extracted with TRIzol (Invitrogen) and used to construct a cDNA library. 5 μg of full-length double-stranded cDNA was then processed by the standard genome sequencer library preparation method using the 454 DNA library preparation kit (titanium chemistry) to generate single-stranded DNA ready for emulsion PCR (emPCRTM). The cDNA library was sequenced according to GS FLX technology (454/Roche Applied Science). Sequence reads were trimmed by excluding adapters and low quality regions using the NGen module of the DNAStar Lasergene software suite. Subsequently, assembly was performed with SeqMan Pro (DNASTAR) using high stringency de novo transcriptome assembly (100% identity between reads with 50-nucleotide sequence overlap). Similar reads were assembled into contigs using CLC Genomics Workbench 3 with its default parameters. All assembled contigs and rest reads were searched against the NCBI subset of the EST database with BLASTn. All of these sequences were also searched with BLASTx in all six reading frames to the non-redundant protein database to determine their correct translation products. For both searches, Blast version 2.2.25+ was used, and an e value threshold of e ≤ 10−3 with a bit score of >40 was considered and recorded as a significant match for each query sequence. We analyzed the Blast results and used a homemade PERL script to classify representative sequences into five categories (“toxin-like,” “putative toxins,” “cellular proteins,” “unknown function,” and “no hit”). Functional characteristics of the transcriptome were predicted using BLAST2GO software (20.Conesa A. Götz S. García-Gómez J.M. Terol J. Talón M. Robles M. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research.Bioinformatics. 2005; 21: 3674-3676Crossref PubMed Scopus (8558) Google Scholar) with the NCBI non-redundant protein database (cut-off e value of ≤10−5) using contigs. Each contig with GI accession (NCBI) of the significant hits retrieved was assigned GO terms according to molecular function, biological process, and cellular component ontologies at a level that provided the most abundant category numbers (21.Ashburner M. Ball C.A. Blake J.A. Botstein D. Butler H. Cherry J.M. Davis A.P. Dolinski K. Dwight S.S. Eppig J.T. Harris M.A. Hill D.P. Issel-Tarver L. Kasarskis A. Lewis S. Matese J.C. Richardson J.E. Ringwald M. Rubin G.M. Sherlock G. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium.Nat. Genet. 2000; 25: 25-29Crossref PubMed Scopus (27012) Google Scholar). The identification of toxin peptide sequences was carried out from the raw data using tBlastn, and a signal peptide can be predicted with the SignalP version 3.0 program (available from the SignalP server). The propeptide cleavage site was ascertained from the known start site of previously characterized mature toxins. As mentioned previously, such long sequence reads are likely to contain the full nucleic sequences of toxin precursors. The toxin-like sequences, the sequences representing no hits, and those with an abundance of cysteine residues may encode new toxin peptides. Toxin precursors were selected out according to the four following parameters: the proteins came from a full open reading frame (ORF) translated by Geneious software (22.Kearse M. Moir R. Wilson A. Stones-Havas S. Cheung M. Sturrock S. Buxton S. Cooper A. Markowitz S. Duran C. Thierer T. Ashton B. Meintjes P. Drummond A. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data.Bioinformatics. 2012; 28: 1647-1649Crossref PubMed Scopus (12577) Google Scholar); the proteins contained more than 4 Cys residues (23.Duan D. Rong M. Zeng Y. Teng X. Zhao Z. Liu B. Tao X. Zhou R. Fan M. Peng C. Chen P. Liang S. Lu M. Electrophysiological characterization of NSCs after differentiation induced by OEC conditioned medium.Acta Neurochir. 2011; 153: 2085-2090Crossref PubMed Scopus (15) Google Scholar); the proteins contained more than 45 amino acids; the toxin precursors were clustered into toxin groups according to their sequence similarity. All precursor sequences were aligned using ClustalX. The resulting alignment was imported into MEGA software to construct phylogenetic tree by the neighbor-joining method (24.Kumar S. Nei M. Dudley J. Tamura K. MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences.Brief. Bioinform. 2008; 9: 299-306Crossref PubMed Scopus (2855) Google Scholar), and bootstrap values were estimated from 500 replicates. Toxins were purified and identified as described previously (10.Tang X. Zhang Y. Hu W. Xu D. Tao H. Yang X. Li Y. Jiang L. Liang S. Molecular diversification of peptide toxins from the tarantula Haplopelma hainanum (Ornithoctonus hainana) venom based on transcriptomic, peptidomic, and genomic analyses.J. Proteome Res. 2010; 9: 2550-2564Crossref PubMed Scopus (83) Google Scholar). Rat DRG neurons were acutely dissociated from 30-day-old Sprague-Dawley rats and maintained in short term primary culture according to the method described by Xiao and Liang (25.Xiao Y. Liang S. Inhibition of neuronal tetrodotoxin-sensitive Na+ channels by two spider toxins: hainantoxin-III and hainantoxin-IV.Eur. J. Pharmacol. 2003; 477: 1-7Crossref PubMed Scopus (62) Google Scholar). Ionic currents were recorded from DRG cells under whole-cell patch clamp techniques using an Axon 700B patch clamp amplifier (Axon Instruments, Irvine, CA) at room temperature (20–25 °C). Patch pipettes with DC resistance of 2–3 megaohms were fabricated from borosilicate glass tubing (VWR micropipettes, VWR Co., West Chester, PA) using a two-stage vertical microelectrode puller (PC-10, Narishige, Tokyo, Japan) and fire-polished by a heater (Narishige). Experimental data were acquired and analyzed using the programs Clampfit version 10.0 (Axon Instruments) and Sigmaplot version 9.0 (Sigma). For Na+ current recording, the pipette solution contained 145 mm CsCl, 4 mm MgCl2, 10 mm HEPES, 10 mm EGTA, 10 glucose mm, 2 mm ATP (PH 7.2), and the external solution contained 145 mm NaCl, 2.5 mm KCl, 1.5 mm CaCl2, 2 mm MgCl2, 10 mm HEPES, 10 mm d-glucose (pH 7.4). For K+ current recording, the pipette solution contained 135 mm KCl, 25 mm KF, 9 mm NaCl, 0.1 mm CaCl2, 1 mm MgCl2, 1 mm EGTA, 10 mm HEPES, and 3 mm ATP-Na2, adjusted to pH 7.4 with 1 m KOH, and the external bath solution contained 150 mm NaCl, 30 mm KCl, 5 mm CaCl2, 4 mm MgCl2, 0.3 mm tetrodotoxin (TTX), 10 mm HEPES, and 10 mm d-glucose, adjusted to pH 7.4 with 1 m NaOH. For Ca2+ current recording, the internal solution contained 110 mm Cs-methane sulfonate, 14 mm phosphocreatine, 10 mm HEPES, 10 mm EGTA, 5 mm ATP-Mg, adjusted to pH 7.3 with CsOH, and the external solution contained 10 mm BaCl2, 125 mm tetraethylammonium chloride, 0.3 mm TTX, and 10 mm HEPES, adjusted to pH 7.4 with tetraethylammonium hydroxide. The mRNAs of six venom glands from the tarantula Ornithoctonus hainana were extracted and sequenced using GS FLX technology (454/Roche Applied Science) following the manufacturer's protocol. Sequencing revealed a total of 249,549 reads (amounting to ∼757 Mb) with an average length of ∼328 bases/read (maximum 830 bp, minimum 40 bp, 6.72% of reads <100 bp, 84.6% of reads 100–500 bp, 8.63% of reads >500 bp). The raw sequencing data can be downloaded from SRA (NCBI) using accession number SRP040123. After removing sequences of low quality, a total of 215,640 reads were assembled into 65,432 contiguous DNA sequences (contigs) with an average length of 625 bp (36.3 reads/contig), with the rest remaining as single reads. Whereas this study focused mainly on toxin peptides, numerous other protein sequences were identified and will be described elsewhere. As outlined under “Experimental Procedures,” we searched for toxin peptide sequences directly from the sequencing reads, because the average read length of >200 bp allowed the identification of full-length toxin precursors. Toxin peptides were also searched for in the contigs, and no additional toxin peptide sequences were found. In total, 52,570 reads displayed similarity to known peptide toxins or toxin-like sequences; the category of putative toxins includes sequences rich in cysteine residues and sharing sequence identity with toxins or proteins including the ICK motif (5%) that were not identified by a BLAST search; the category of cellular proteins includes transcripts coding for proteins involved in cellular processes (44%); the unknown function category includes reads that shared sequence identity with previously described sequences with no functional assessment or hypothetical genes; and the no hit category indicates no match with currently known sequences. The results are summarized in Fig. 1. A search against publicly available databases (nr/NCBI, Swiss-Prot + TREMBL/EMBL) revealed that 8,773 high confidence proteins were associated with GO terms and further grouped into the categories of molecular functions, biological processes, and cellular components at the second level according to standard gene ontology terms. Based on annotations from GO analysis (Fig. 2), transcripts were categorized into 2,610 groups with 80% identity threshold. These included functional annotations for 816 biological process (BP), 798 molecular function (MF), and 996 cellular component (CC) categories (Fig. 2). Highly expressed transcripts were enriched in metabolism and translation processes, indicating that venom glands are metabolically active and engaging in the intensive protein synthesis and processing required for venom production. Transcripts associated with binding, catalysis, and channel regulation were also highly represented, indicating that venom peptides may play an important role in prey inactivation by binding to ion channels. GO terms related to redox homeostasis and proteolysis were also enriched, which may be related to the extensive post-translational modification of spider toxins that was reported previously (9.Chen J. Zhao L. Jiang L. Meng E. Zhang Y. Xiong X. Liang S. Transcriptome analysis revealed novel possible venom components and cellular processes of the tarantula Chilobrachys jingzhao venom gland.Toxicon. 2008; 52: 794-806Crossref PubMed Scopus (36) Google Scholar). Although spider toxins contain diverse disulfide bridge patterns and fold into a variety of three-dimensional structures, cysteine-rich domains are a common feature shared by many toxin sequences (26.Naamati G. Askenazi M. Linial M. ClanTox: a classifier of short animal toxins.Nucleic Acids Res. 2009; 37: W363-W368Crossref PubMed Scopus (49) Google Scholar, 27.Kuzmenkov A.I. Fedorova I.M. Vassilevski A.A. Grishin E.V. Cysteine-rich toxins from Lachesana tarabaevi spider venom with amphiphilic C-terminal segments.Biochim. Biophys. Acta. 2013; 1828: 724-731Crossref PubMed Scopus (27) Google Scholar). Overall, 1,136 toxin precursors were identified in the venom gland transcriptome, and 65.8% of the mature peptides included two adjacent cysteine residues. From previous proteomics results (10.Tang X. Zhang Y. Hu W. Xu D. Tao H. Yang X. Li Y. Jiang L. Liang S. Molecular diversification of peptide toxins from the tarantula Haplopelma hainanum (Ornithoctonus hainana) venom based on transcriptomic, peptidomic, and genomic analyses.J. Proteome Res. 2010; 9: 2550-2564Crossref PubMed Scopus (83) Google Scholar), we were able to predict precursor endoproteolytic and amidation sites with high confidence, and 90 toxins and variants accounted for 95% of all toxin precursors. These were divided into 14 classes (classes A–N) based on the number of cysteine residues present (Table 1), and 70 sequences did not fall into these categories in class O (Fig. 3). Of the 18 H. hainanum peptide toxins characterized previously, 11 belonged to class G along with 252 variants, four belonged to class E along with 39 variants, and the other three belonged to classes C, L, and M along with 19, 25, and 4 variants, respectively.TABLE 1The main features of H. hainanum venom gland toxins Open table in a new tab FIGURE 3.EST processing, toxin identification, and classification.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Class A contained seven novel transcripts, including HN-Aa and its variants (Fig. 4). The signal peptide and propeptide of these precursors are identical, and their cleavage sites are LFA/ED and LESEK, respectively. The mature peptide has 12 Cys residues, which is the highest number in a toxin peptide scaffold in H. hainanum reported to date, although two other spider toxins share this cysteine pattern (28.Zhang Y. Chen J. Tang X. Wang F. Jiang L. Xiong X. Wang M. Rong M. Liu Z. Liang S. Transcriptome analysis of the venom glands of the Chinese wolf spider Lycosa singoriensis.Zoology. 2010; 113: 10-18Crossref PubMed Scopus (66) Google Scholar, 29.Bohlen C.J. Priel A. Zhou S. King D. Siemens J. Julius D. A bivalent tarantula toxin activates the capsaicin receptor, TRPV1, by targeting the outer pore domain.Cell. 2010; 141: 834-845Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). TX-L precursors from Lycosa singoriensis superfamily VI have the cysteines arranged C-CC-C-C-C-C-C-C-C-C-C, whereas HN-Aa has the double cysteine shifted to the left. The double knot toxin from the earth tiger tarantula (Selenocosmia huwena) has the pattern C-C-CC-C-C-C-C-CC-C-C, and this sequence shares 87% similarity with HN-Aa. Double knot toxin selectively and irreversibly activates the capsaicin- and heat-sensitive channel TRPV1 (29.Bohlen C.J. Priel A. Zhou S. King D. Siemens J. Julius D. A bivalent tarantula toxin activates the capsaicin receptor, TRPV1, by targeting the outer pore domain.Cell. 2010; 141: 834-845Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). This cysteine pattern therefore indicates that HN-Aa may be a TRPV1 channel activator. The six variants identified showed high similarity with the mature peptide, and the presence of different lengths of mature peptide results in great potential for toxin variability. Class B contains only four members, the fewest of all classes (Fig. 4). HN-Ba and its three variants include 11 Cys residues, and HN-Ba precursors contain over 100 residues, which includes the consensus VIAYA cleavage signal, whereas the propeptide numbered only four residues. A large number of Gln residues were found in the mature peptide, and the C terminus is rich in Thr and Ser residues, which is strikingly different from other toxins present in this species. The cysteine pattern shares homology with the U1-hexatoxin-Hsp201a from the funnel web spider (30.Wen S. Wilson D.T. Kuruppu S. Korsinczky M.L. Hedrick J. Pang L. Szeto T. Hodgson W.C. Alewood P.F. Nicholson G.M. Discovery of an MIT-like atracotoxin family: spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins.Peptides. 2005; 26: 2412-2426Crossref PubMed Scopus (30) Google Scholar). Other than this highly conserved cysteine pattern, HN-Ba showed no obvious sequence similarity with other peptide toxins. Class C contains 29 transcripts that clustered into the known toxin HN-Ca (HNTX-XIV) and two novel toxins (HN-Cb and HN-Cc; Fig. 4), all of which included 10 Cys residues, but class C toxins were further divided into two clades based on cysteine pattern. HN-Ca and HN-Cb shared the pattern -C-C-CC-C-C-C-C-C-C- and formed the first clade, and these toxins do not have a propeptide but do share a signal peptide containing the cleavage site ELVSC. The mature HN-Cb peptide is rich in Val residues and has a C terminus that is ∼20 residues longer than HN-Ca (HNTX-XIV), indicating diverse functions. Further research is needed to investigate whether these additional residues are cleaved during post-translational modification or remain and have some additional functional significance. HN-Cc exhibited the cysteine pattern -C-C-C-CC-C-C-C-C-C-, and the signal peptide, propeptide, and mature peptide were distinct from HN-C[a∼b], although HN-Cc peptides shared sequence similarity and included an extended cysteine variant not present in HNTX-XVIII. The four toxin precursors in cla" @default.
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• W2093034951 title "Structural and Functional Diversity of Peptide Toxins from Tarantula Haplopelma hainanum (Ornithoctonus hainana) Venom Revealed by Transcriptomic, Peptidomic, and Patch Clamp Approaches" @default.
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